Post-translational Modifications Required for Coagulation Factor Secretion and Function

Kaufman, Randal J.

doi:10.1055/s-0037-1615018

Cited by 113 publications

(126 citation statements)

References 133 publications

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“…[47][48][49] In addition to demonstrating the biologic activity of muscle-synthesized protein, however, we wanted to conduct detailed biochemical characterization because the nature of the posttranslational modifications can influence the recovery of the protein 28 and, theoretically, the immunogenicity. 50 Short of isolating muscle-synthesized F.IX from the plasma of treated human subjects, there is no straightforward method for obtaining F.IX synthesized in skeletal muscle because it is not possible to maintain mature myocytes in culture.…”

Section: Discussionmentioning

confidence: 99%

“…Sites of O-linked glycosylation within liversynthesized F.IX have been identified at residues Ser 53, Ser 61, 28,41 but the function of these residues remains unknown. The O-linked sites are not easily analyzed by enzymatic release because O-glycanase digestion requires an ␣-GalNAc linkage, nor are they readily examined using oligosaccharide profiling because enzymatic removal and fluorescent labeling of O-linked residues are inefficient processes that require large amounts of starting material to yield detectable signal.…”

Section: Carbohydrate Analysismentioning

confidence: 99%

“…It undergoes extensive posttranslational modification, including cleavage and removal of the pre-pro leader sequence of 46 amino acids; ␥-carboxylation of the first 12 glutamic acid residues; partial ␤-hydroxylation of Asp 64; N-linked glycosylation at Asn 157 and Asn 167; O-linked glycosylation at Ser 63, Ser 61, and Thr 159, 169, 172, and 179; sulfation of Tyr 155; and phosphorylation at Ser 158. 27,28 Whether the modifications critical for biologic activity are carried out efficiently in human muscle has not been determined.…”

Section: Introductionmentioning

confidence: 99%

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Posttranslational modifications of recombinant myotube-synthesized human factor IX

Arruda

Hagstrom

Deitch

et al. 2001

Blood

116

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Recent data demonstrate that the introduction into skeletal muscle of an adenoassociated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical ␥-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity.Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and ␥-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX. (Blood. 2001;97:130-138)

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Section: Discussionmentioning

confidence: 99%

Section: Carbohydrate Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Posttranslational modifications of recombinant myotube-synthesized human factor IX

Arruda

Hagstrom

Deitch

et al. 2001

Blood

116

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“…When we started those experiments, mutations in that FVIII region were known to be associated with a high incidence of inhibitors in mild/moderate haemophilia A patients [10], although the reason for such an association was unclear. Moreover, inhibitor antibodies recognizing the C1 domain had never been demonstrated and no role of the latter domain in FVIII function and/or stability had been determined [11,12].…”

Section: Introductionmentioning

confidence: 99%

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody

Jacquemin

2010

Haemophilia

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Summary. The mechanism of action of antibodies inhibiting partially factor VIII (FVIII) activity (type II inhibitor) is still poorly understood. We produced an unusual type II monoclonal antibody, called LE2E9, derived from a patient with mild haemophilia A. The antibody displayed several unexpected structural and functional properties such as glycosylation in the variable region, binding to the FVIII C1 domain, inhibition of maximum 80-90% FVIII activity when in excess over FVIII, and prevention of FVIII binding to von Willebrand factor (VWF). Those unusual characteristics of the antibody prompted multidisciplinary studies to determine its mechanism of action and the role of the FVIII C1 domain. Enzymatic deglycosylation and site-directed mutagenesis indicated that the oligosaccharides do not determine the affinity of the antibody but enhanced its FVIII neutralizing activity. Modification of glycosylation in the variable region of antibodies therefore contributes to the diversity of FVIII type II inhibition and provides a novel strategy with which to modulate the functional activity of antibodies. Investigation of the FVIII C1 domain function led to identification of mutations located in that domain and impairing FVIII binding to VWF as a common cause of mild/moderate haemophilia A. Finally, the cloning of human monoclonal antibodies inhibiting partially FVIII activity opened the way to evaluate such antibodies as a novel type of anticoagulant drug. This concept was validated in animal models of thrombosis. Those studies are expected to have a significant impact on the optimization of treatments for patients with inhibitor as well as for patients with thrombophilia.

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“…g-Carboxylation and b-hydroxylation are PTMs characteristic of a small number of proteins, mainly a subset of proteins involved in blood coagulation [14,15]. These modifications are undertaken by specific carboxylase and hydroxylase enzymes, with conversion of target glutamate residues in the protein backbone into g-carboxyglutamate (Glu !…”

Section: G-carboxylation and B-hydroxylationmentioning

confidence: 99%

Post‐Translational Modifications in the Context of Therapeutic Proteins: An Introductory Overview

Walsh

2009

Post‐translational Modification of Protein Biopharmaceuticals

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Post-translational Modifications Required for Coagulation Factor Secretion and Function

Cited by 113 publications

References 133 publications

Posttranslational modifications of recombinant myotube-synthesized human factor IX

Posttranslational modifications of recombinant myotube-synthesized human factor IX

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody

Post‐Translational Modifications in the Context of Therapeutic Proteins: An Introductory Overview

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